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omgmbd > L7: Cell Culture > Flashcards

L7: Cell Culture Flashcards

1
Q

What are advantages of cell cultures?

A

→Control of the physiochemical environment (pH, temperature, osmolarity) and physiological conditions (levels of hormones and nutrients)

→Control of the micro-environment of the cells

→Cells can be easily characterised by cytological or immune-staining techniques and visualised using imaging techniques

→Cells can be stored in liquid nitrogen for long periods (cryopreservation)

→Cells can be easily quantified
Reduces use of animals in scientific experiments

→Cheaper to maintain

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2
Q

What are the two types of cell cultures?

A

→primary tissue cells

→immortalised cell lines

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3
Q

What are the differences between primary tissue cells and cell line culture?

A

→primary tissue cells have limited lifespan retain cells
but CL has infinite lifespan and loses specificity
→pre-characterised and ready to use but CL requires authentication required before use
→study cells with varied donor characteristics but CL study single donor repeatedly
→PTC are less heterogenous

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4
Q

What are the characteristics of PTC?

A

→Cells derived directly from tissues/patients (unmodified)
→Finite lifespan (~6-7 divisions)

→Cells divide and/or differentiate

→Cells carry out normal functions

→Living conditions similar to body environment

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5
Q

What are the two methods for isolation of cells?

A

→explant culture-Cells allowed to migrate out of an explant
→Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)

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6
Q

Why are haemotopoeic cells an exception when isolating cells?

A

→Do not need to be disaggregated
→already are as individual cells circulating in blood.
→Use density centrifugation instead

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7
Q

Why is centrifugation used for haematopoietic cells?

A

→Takes advantage of the different densities of diff blood cells

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8
Q

Which cells are densest?

A

→Granulocytes

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9
Q

What are other methods of isolation?

A

→Immuno-purification- using antibody-coated magnetic beads

→Fluorescence activated cell sorter

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10
Q

What are the non-haematopoietic cells?

A 
→liver
→muscle
→endothelial
→skin
→nerves
→ fibroblasts
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11
Q

What are the disadvantages of primary tissue cells?

A 
→Inter-patient variation
→Limited number (small amount at high cost)
→Finite lifespan and hard to maintain
→Difficult molecular manipulation
→Phenotypic instability
→Variable contamination
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12
Q

What are the characteristics of cell lines?

A

→Immortalised cells from one type of primary tissue cells

→Less limited number of cell divisions (~30) or unlimited

→Phenotypically stable, defined population

→Limitless availability
→Easy to grow
Good reproducibility

→Good model for basic science if same cell lines are used

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13
Q

What are the two types cell lines?

A
  1. Isolated from cancerous tissues (e.g. HeLa cells)

2. Immortalisation of healthy primary cultures (usually through genetic manipulation)

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14
Q

What are the targets in cells for production of cell lines through genetic manipulation?

A

→processes that regulate cellular growth and ageing

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15
Q

What are process that regulate cellular growth and ageing?

A

→p53
→pRB
→telomerase

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16
Q

What happens as telomeres shorten?

A

→cell division stops

→ Apoptosis (p53, pRb)

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17
Q

How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?

A

→taking advantage of viral ‘oncoproteins

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18
Q

What are the viruses used to alter a cell’s capability for its finite number of divisions and their viral oncoproteins?

A

→Simian Virus-40
(SV40)- Large T antigen, Small t antigen
→Human Papilloma
Virus (HPV), E6 E7

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19
Q

What are the targets for viruses used in cell line division numbers?

A

→p53

→pRb

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20
Q

How does SV40 T-antigen work in cell line generation?

A

→interacts with p53 and pRb

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21
Q

What does use of SV40 T-antigen in cell line allow?

A

→increased growth without loss of function of these proteins

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22
Q

What does E6 and E7do?

A

→targets p53 for degradation, and E7 binds to pRb inactivating it

23
Q

What is the phenotype like in cell lines made using E6 and E7?

A

→maintain a differentiated phenotype

24
Q

What do some cells need in generating cell lines?

A

→need both introduction of the telomerase gene

→inactivation of the pRb/p53 for “immortalisation”

25
Q

Are TERT enzymes active in somatic cells?

A

→no

26
Q

How is TERT activated in somatic cells?

A

→need to be transfected
→Make a vector containing selection marker eg neomycin(antibiotic resistance) and telomerase gene.

→Plasmid has to be circularised

→Treat cells with antibiotic and only cells with it will survive

27
Q

What are the advantages of 2D cell culture?

A

Simple, well established

- Affordable

28
Q

What are the disadvantages of 2D cell cultures?

A

→Forced apical-basal polarity
→High stiffness
→Limited communication with other cells
→No diffusion of gradients
→Results not relevant to human physiology
→Cells lose their original phenotype and functional characteristics

29
Q

What are the advantages of 3D cultures?

A

→Adhesion in all three dimensions
→No forced polarity
→ Variable stiffness
→ Diffusion gradients of nutrients and waste products
→More relevant to human physiology
→Allow cells to aggregate- balls of cells

30
Q

What are the disadvantages of 3D cultures?

A

→More complex

→Added expense

31
Q

What are the two types of shapes of 3D cultures?

A

→spheroids

→organoids

32
Q

Describe spheroids

A

→3D
→cellular aggregate composed of 1(or more) cell types that grow and proliferation
→exhibit enhanced physiological responses
→do not differentiate or self-organise

33
Q

Describe organoids

A 
→3D culture
→derived from PSCs, AdSC
→ cells spontaneously self organise into properly differentiated functional cells types
→ resemble in vivo counterpart
→resemble some function of organ
34
Q

What do patient derived organoids allow?

A

→the study cancer drug resistance

→Make treatment based on results of the treatment on culture

35
Q

Why are patient-derived cells organoids?

A

→derived from primary tissue cells

36
Q

What is the percentage mimicry of phenotype in organoids?

A

→85%

37
Q

Define transefection

A

process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab.

38
Q

Give examples of non-viral transfection methods

A

→plasmid,

→ CRISPR/Cas9 complex

39
Q

Give examples of chemical transfection

A

→Lipofection
→Calcium phosphate
→Cationic polymer
→Magnet-mediated transfection

40
Q

Why is transfection performed in 2D?

A

→if 3D not all cells would be transfected

41
Q

Give examples of physical transfection

A 
→Electroporation
→Nucleofection
→Microinjection
→Laserfection/ optoinjection
→Biolistic Particle Delivery
42
Q

What is lipofection?

A

→Using cationic lipid transfection systems

→uses liposomes

43
Q

Why are liposomes used for lipofection?

A

→has unilamellar liposomal structure
→has phospholipid bilayer
→net positive charge and the DNA/plasmid will have a negative charge

44
Q

Describe lipofection

A 
1. Interaction with 
the cell
membrane
2. Taken up by 
endocytosis
3. Release from the 
endosome
→4. Transport to the
nucleus
→5. Entry to the nucleus inefficient 
and may need mitosis
45
Q

Do liposomes deliver hydrophobic or hydrophilic drugs?

A

→hydrophobic or hydrophilic

46
Q

How do you ensure the drug is delivered into the tissue of interest?

A

→Attach tissue specific antigens to the liposomes

47
Q

How is electroporation carried out?

A

→High electric field
→Forms pores- increase permeability
→DNA goes through cell
which then reseal

48
Q

What does the rate of pore sealing in electroporation dependent on?

A

→temperature

49
Q

What are the characteristics of nucleofection?

A

→Combination of electroporation and lipofection

  • Increased efficiency particularly of non-dividing cells
  • Technology is protected under patent
  • Different solution and protocols are used for each cell type
50
Q

Describe viral infection

A 
1. plate HEK293T cells
→transfect plasmids
→collect 1st supernatant
→refrigerate
→collect 2nd supernatant
→collect viral pellet after centrifuging
→use for transduction
51
Q

What is HEK293T?

A

→used as vehicle for production of material in viral transfection

52
Q

What types of cells are used in viral infection?

A

→Retrovirus,
→Adenovirus,
→Lentivirus commonly
are used.

53
Q

What enables HEK293T cells to produce recombinant proteins within plasmid vectors?

A

→contains the SV40 large T antigen

omgmbd (29 decks)

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